Method for the determination of the formation of endothelins for medical diagnostic purposes, and antibodies and kits for carrying out such a method

ABSTRACT

An in vitro method for the determination of the formation of endothelins in serious diseases, in particular cardiovascular diseases, inflammations, sepsis and cancer, in whole blood, plasma or serum of a human patient for medical diagnostics purposes, in which relatively long-lived peptide fragments of the processed primary prepro- or proendothelins are determined which contain neither the actual biologically active endothelin nor its direct precursor big endothelin, in particular a C-terminal peptide fragment.

The invention relates to methods for the determination of the formationof endothelins in serious diseases by determining peptide fragments ofthe corresponding proendothelin, in particular a relatively long-livedC-terminal partial peptide of preproendothelin-1, in the circulation(whole blood, plasma or serum) for medical diagnostic purposes, inparticular in sepsis diagnosis, in cardiac diagnosis and, for example,also in cancer diagnosis and/or generally in the diagnosis of thosepathological conditions in which endothelins play an important role forthe course of the disease.

Where simply the term “endothelin” is used in the present Application,this term primarily represents endothelin-1 (ET-1). However, acorresponding statement is frequently also applicable to other isoformsof endothelins, and therefore a limitation to endothelin-1 frequentlyappears unnecessary and the invention is also intended to include otherendothelins in a wider sense.

In this description, the term “diagnostic” is used in principle as asimplifying general term which is intended to include in particularprognosis/early prognosis and therapy-accompanying monitoring.

The determinations are effected in particular by means of specificimmunodiagnostic methods, in particular by means of immunoassays of atype in which at least one marked antibody is employed (sandwich assay;competitive assay, e.g. according to the SPALT or SPALT principle).

Endothelin-1 (ET-1), a peptide comprising 21 amino acids, is thestrongest known vasoconstrictor. Since its discovery in 1988 byYanagisawa et al. [27; numerical data in square brackets relate to theattached list of references], biosynthesis, mode of action andassociation with diseases have been comprehensively investigated andsummarized in topical review articles [1, 7, 12, 17, 24]. There arethree isoforms of endothelin which are coded by different genes(endothelin-1, endothelin-2, endothelin-3) of which endothelin-1 ispresent in the greatest concentrations and is the most effective.Endothelin-1 is synthesized in endothelium cells, in the lung, in theheart, in the kidney and in the brain. The primary translation productof the human endothelin-1 gene is a peptide comprising 212 amino acids,preproendothelin-1 (SEQ ID NO:1). In the secretion process, a shortN-terminal signal sequence (amino acids 1-17) of the preproendothelin isremoved by the signal peptidase. The proendothelin obtained is thenprocessed by the protease furin on dibasic amino acid pairs to give abiologically inactive peptide comprising 38 amino acids, big endothelin(SEQ ID NO:3), from which finally the mature, biologically activeendothelin-1 (SEQ ID NO:2) is formed by means of endothelin-convertingenzymes (ECEs). Endothelin acts via the bond to specific receptors whichare localized on muscle cells, myocytes and fibroblasts. This bond leadsto efflux of calcium, activation of phospholipase C and inhibition ofNa/K ATPase. In addition to the vasoconstrictive effect, endothelin alsohas growth-regulating properties.

In view of the detectable and presumably numerous and seriousphysiological effects of endothelins, in particular endothelin-1,various assays for its immunodiagnostic determination have beendeveloped since the time of its identification and have been used formeasurements of endothelin(s), in particular in human plasmas. Theresults of such determinations are the subject of numerous publications.

Raised plasma concentrations of endothelin-1 and big endothelin havebeen described for various clinical pictures [17]. These includecardiovascular diseases [1] (inter alia pulmonary hypertension [21],atherosclerosis [13], congestive heart failure [25], myocardialinfarction [20]), sepsis and septic shock [11, 22, 23], cancer [2, 3,15, 18], etc.

The immunoassays used for the measurements of endothelins in plasmasamples (cf. the review in [17]) belonged in particular to theradioimmunoassay type (with marked endothelin-1 as competitor) or to theEIA/ELISA type and aimed exclusively at the determination of endothelinor the determination of an endothelin immunoreactivity. Assays of theRIA type have low specificity and also determine related peptidescontaining the endothelin sequence.

However, it was found that endothelin-1 (ET-1) has an extremely shortresidence time in the circulation and that it is removed from thecirculation after only 1-2 min [6]. Since endothelin-1 in blood andplasma is considered to be stable [6], its distribution in other tissueand its rapid and high-affinity bonding to receptors are regarded as themost important reason for the short residence time. In certain tissuesand body fluids, substantially higher endothelin-1 concentrations than,for example, in plasma could consequently be determined [1, 7]. In viewof these circumstances, serious doubt was cast on the validity of thedetermination of ET-1 in plasma samples [17]. It is in fact to beassumed that the instantaneous ET-1 concentrations determinable in aplasma sample and reflecting in certain circumstances only a transitionstate are not important for the physiological effects of endothelin(ET-1) but that the sum of all free and bound, e.g. tissue- andreceptor-bound, physiological ET-1 concentrations present in theorganism are of much greater relevance.

The determination of the ET-1 precursor, of so-called big endothelin(“big ET-1”; SEQ ID NO:3), has the advantage over the determination ofET-1 that the residence time of “big ET-1” in the circulation issubstantially longer than that of the ET-1 liberated therefrom. In anumber of investigations, this “big endothelin” was therefore determinedinstead of the actual endothelin. In particular assays of the sandwichtype which permit a reliable distinction of big endothelin-1 fromprocessed ET-1 and other endothelins were used for its specificdetermination [4, 8, 10].

They showed that, in certain diseases, the increased ETimmunoreactivities measured can be attributed to big ET.

The selective measurement of big-ET-1 represents only a gradualimprovement but not an actual solution to the problem, since bigendothelin too can be processed rapidly in blood circulation to giveendothelin [1, 5, 9]. It therefore likewise has a relatively shortbiological half-life (20-25 minutes) [10], and consequently a measuredvalue of the big endothelin determinable in the plasma likewiserepresents only an instantaneous plasma concentration and does notreflect the actual physiologically effective concentrations ofendothelin. ET-1 formed physiologically under the conditions of adisease but already processed and bound into tissues or to receptors isnot detected in plasma in the determination of big-ET-1. The totalamount of physiologically active endothelin is therefore alsounderestimated in a measurement of big endothelin. An attempt to performa supplementary specific measurement of the C-terminal peptide fragmentof big-ET-1 (with the amino acids 74-90 of preproendothelin or the aminoacids 20-38 of big endothelin) formed in the enzymatic cleavage ofbig-ET-1 in addition to ET-1 showed that this peptide is even lessstable than ET-1 and is therefore unsuitable for measurements [10].

The prior art discloses only one commercial competitive test (N-terminalrange 18-50, commercially available from Phoenix Pharmaceuticals; usefor the sepsis diagnosis described in WO 00/22439) for evaluating rangesof proendothelin outside that of big endothelin. No information has beenpublished regarding the stability and nature of the analyte to beevaluated using this assay.

It is the object of the present invention to develop an assay methodwhich reflects the endogenous formation of big endothelin andendothelin, i.e. the total physiological concentration and hence actionof endothelin, more reliably than the determinations to date of ET orbig ET in plasma.

Such a method should be valid and capable of routine use and should becapable of providing reliable values for the physiological production ofET (ET-1) and/or its precursors in various pathological conditions, inparticular in sepsis or other pathological conditions in which increasedvalues for endothelin play a role.

This object is achieved, according to the invention, by determining notET or big ET but a comparatively long-lived prepro- or proendothelinpartial peptide which does not contain the ET or big ET sequences, inparticular a C-terminal partial peptide which contains at least theamino acids 168-212 of pre-proET-1, in a whole blood, plasma or serumsample of a human patient, for diagnostic purposes.

Claim 1 relates to the teaching of the present invention. Advantageousand currently preferred embodiments of the invention are described inthe subclaims.

The invention is based on experimental investigations by the Applicantin which said Applicant was able to show that those parts ofpreproendothelin which do not represent direct precursors of endothelincomprise long-lived peptides which are suitable for measuring purposesand can be measured in blood samples reliably and with a high clinicalrelevance.

Endothelin-1 is formed physiologically by processing of the largerprecursor molecule preproendothelin (SEQ ID NO:1) or of the secretedproendothelin obtained therefrom. During such processing, furtherpeptides form in primarily stoichiometric amounts in addition to bigendothelin (and therefrom endothelin), which further peptides, however,have to date not been the subject of scientific investigations and aboutwhose possible further processing and stability nothing has beendisclosed to date. At the beginning of the investigations by theApplicant, it was hoped that it would be possible to show that at leastone of the hypothetical further peptide cleavage products is present inblood samples (whole blood, plasma or serum samples) and would prove tobe relatively stable and which cleavage product might therefore besuitable to serve as a measure of the physiological formation ofendothelins independently of an endothelin concentration actuallymeasurable in plasma.

The evaluation of such a cleavage product might therefore represent themethod sought for the determination of the physiological endothelinconcentration or production which is designated in the Claims asdetermination of the “formation of endothelins”. This term is used torefer to the fact that—assuming only one route of formation, namely thesingle known route of formation, of endothelin-1 from proendothelin—thephysiological concentrations of endothelin 1 which are formed inassociation with the disease can correspond only to the amount of thepreviously processed preproendothelin or proendothelin. If the partialpeptides formed in the same stoichiometric concentration in addition tobig endothelin or endothelin are stable “metabolic waste products” whichare neither bound to receptors nor distributed in tissues, they must bepresent in the circulation. Without wishing thereby necessarily to implya certain physiological mechanism, the “determination or measurement ofthe formation of endothelins” can therefore also be regarded asmeasurement “of the secretory activity” or of the “secretoryproendothelin production”.

In this Application, the peptide fragments to be determined arecharacterized as “long-lived”. This term means that the residence timeof the peptide fragment to be determined in the circulation (in wholeblood) is considerably longer than that of endothelin or of bigendothelin fragments. In particular, “long-lived” means that suchpeptide fragments in whole blood or a plasma obtained therefrom are notsubject to further rapid proteolytic cleavage and, compared with therate of binding of endothelin to receptors and of proteolytic cleavageof cleavable fragments, are removed at a substantially slower rate fromthe circulation or the metabolism.

Owing to said longer stability or “long-lived character”, in thepresence of such fragments the information relating to the alreadyelapsed secretory activities is stored for a period which is suitable atleast for an unproblematic measurement. If it is assumed, for example,that the endothelin precursors are liberated in a single short-termsecretion, the amount of “long-lived” fragments which are measurableafter a certain time corresponds to the originally secreted amount,reduced only by an amount which is linked to the physiological half-lifeof the peptide fragment to be measured in the circulation. If on theother hand, for example, a more or less continuous production of theendothelin precursor during the pathological process is assumed, theformer physiological production of the precursor is cumulativelyreflected in the measurable concentration of a peptide fragment which islong-lived in the above sense, once again reduced only by theconcentration reduction of the peptide fragment which has taken place inthe same period, in accordance with its physiological clearance rate.The active endothelin or its precursor big endothelin may have long beenprocessed or removed from the circulation in the same period and, forexample, may be bound to receptors and therefore no longer measurable.The longer lived a peptide fragment is or the lower its clearance rate,the smaller is the influence of the time of measurement on thecorrectness of the determination of the abovementioned “formation” of abiomarker, i.e. of endothelin. A concentration constant over arelatively long period means in this context that formation andclearance hold the balance. If the concentration decreases, this mayindicate that the secretion of the precursor molecule (for example ofproendothelin) has ceased, for example because the molecular reservoirsare exhausted, and the concentration changes to be observed aredetermined only by the clearance rate.

The results of the measurement of a long-lived peptide fragment withoutknown physiological function thus provides both quantitatively andqualitatively different results from a measurement of a fairlyshort-lived active peptide or its likewise relatively short-livedprecursor.

The investigations by the Applicant which are described in more detailbelow showed that the approach described above gives fruitful results inthe case of the determination of the formation of endothelins.

The investigations carried out and the most significant results of theinvestigations are explained more exactly below, reference being made tofigures. In the figures:

FIG. 1 shows a typical standard curve for the currently preferredsandwich assay with two antibodies which bind to amino acid sequenceswhich correspond to the positions 168-181 and 200-212 ofpreproendothelin-1, for the determination of a C-terminal proendothelinpeptide sequence in human plasma, said sandwich assay being describedmore exactly in the experimental section;

FIG. 2 shows a diagram which shows that, on storage of EDTA plasmasamples from septic and cardiological patients at room temperature over12 hours, no significant loss of immunoreactivity occurs in an assayaccording to FIG. 1;

FIG. 3 a shows the measurement of plasmas of 5 groups of human patientswith different diseases/diagnoses, compared with the measurements forapparently healthy persons; the dotted line indicates the maximum valuefound in healthy persons (line for 100% specificity, based on healthycontrols);

FIG. 3 b shows a diagram corresponding to FIG. 3 a, for four furthergroups of patient plasmas.

The method according to the invention relates in its most general aspectto the determination of a relatively long-lived peptide fragment ofproendothelin-1 which does not contain the amino acid sequences ofendothelin-1 or its precursor big endothelin, in whole blood, plasma orserum samples, i.e. in the circulation of patients, for the indirectdetermination of the formation of endothelins, in particular ofendothelin-1, in serious diseases. According to a preferred embodiment,the peptide fragment determined is a C-terminal fragment to which twoantibodies bind which bind to peptides having amino acid sequences whichcorrespond to the positions 168-181 and 200-212 of preproendothelin-1.

For the practical implementation of the invention, noncompetitivesandwich assays, for example of the type as used for the morefar-reaching detailed investigations and described more exactly below,are particularly preferably provided.

Compared with competitive immunoassays, noncompetitive sandwichimmunoassays (two-sided immunoassays) have a number of advantages, whichinclude the fact that they can be better designed than solid-phaseassays (heterogeneous assays), may be more rugged in terms of handling,can give measured results with a higher sensitivity and are also moresuitable for automation and series measurement. Moreover, they can alsoprovide additional information compared with competitive immunoassayswhich operate with only one type of antibody, in that sandwichimmunoassays recognize only those molecules or peptides with which bothbinding sites for the antibodies used in the sandwich formation arepresent on the same molecule.

The antibodies which may be used may in principle be any desiredsuitable monoclonal and/or polyclonal antibodies, but affinity-purifiedpolyclonal antibodies are currently preferred.

Particularly preferably, the antibodies are obtained by immunization ofan animal, in particular of a sheep, with an antigen which contains asynthetic peptide sequence which corresponds to a short amino acidsequence of preproendothelin-1 and an additional cysteine residue at theN-terminus. In the following experimental section, in particularantibodies which bind to the amino acid sequences 161-181 and 200-212,and their use in an assay are described. However, in the course of theinvestigations, additional antibodies which accordingly bind to thepositions 184-203 and 136-148 were also used. The additional resultsobtained with these further antibodies in measurements are discussedonly globally in this Application.

In a preferred embodiment, the method is carried out as a heterogeneoussandwich immunoassay, in which one of the antibodies is immobilized onany desired solid phase, for example the walls of coated test tubes(e.g. of polystyrene; “coated tubes”; CT) or on microtiter plates, forexample of polystyrene, or on particles, for example magnetic particles,while the other antibody carries a residue which represents a directlydetectable label or permits selective linkage to a label and serves fordetecting the sandwich structures formed. Delayed or subsequentimmobilization with the use of suitable solid phases is also possible.

In principle, all marking techniques which can be used in assays of thetype described may be employed, including marking with radioisotopes,enzymes, fluorescent, chemoluminescent or bioilluminescent labels anddirectly optically detectable color markers, such as, for example, goldatoms and dye particles, as are used, in particular for so-calledpoint-of-care (POC) or accelerated tests for determination in wholeblood samples. In the case of heterogeneous sandwich immunoassays, thetwo antibodies may also have parts of a detection system of the typedescribed below in relation to homogeneous assays.

It is therefore within the scope of the present invention also to designthe method according to the invention as an accelerated test.

The method according to the invention can furthermore be designed as ahomogeneous method in which the sandwich complexes formed from the twoantibodies and the peptide fragment to be detected remain suspended inthe liquid phase. In such a case, it is preferable to mark bothantibodies with parts of a detection system which permits signalgeneration or signal triggering when both antibodies are integrated intoa single sandwich. Such techniques can be designed in particular asfluorescence amplification or fluorescence extinction assays. Aparticularly preferred method of this type relates to the use ofdetection reagents to be used in pairs, as described, for example, inUS-A-4 822 733, EP-B1-180 492 or EP-B1-539 477 and the prior art citedtherein. They permit a measurement which selectively detects onlyreaction products which contain both marking components in a singleimmune complex, directly in the reaction mixture. The technology whichis available under the brands TRACE (Time Resolved Amplified CryptateEmission) and KRYPTOR® and which implements the teachings of theabove-mentioned application may be referred to as an example.

In the investigations by the Applicant, it was found that thedetermination, according to the invention, of the C-terminal peptidefragment of preproendothelin-1 gives highly interesting and relevantresults. As will be shown below, this statement applies not only to thesepsis diagnosis but also to cardiac diagnosis and cancer diagnosis.

It is furthermore assumed that the assays according to the invention canalso be particularly advantageously carried out as part of a so-calledmultiparameter diagnosis, in particular both in the area of cardiacdiagnosis and in sepsis and cancer diagnosis. Further parametersdetermined thereby are, for example, the cardiac parameters ANP, BNP,proANP, proADM and proBNP or sepsis parameters which are selected, forexample, from the group which consists of anti-ganglioside antibodies,the proteins procalcitonin, CA 125, CA 19-9, S100B, S100A proteins,LASP-1, soluble cytokeratin fragments, in particular CYFRA 21, TPSand/or soluble cytokeratin-1 fragments (sCY1F), the peptides inflamminand CHP, other peptide prohormones, glycine-N-acyltransferase (GNAT),carbamoylphosphate synthetase 1 (CPS 1) and C-reactive protein (CRP) orfragments thereof. In said multiparameter assays, it is intended todetermine the measured results for a plurality of parameterssimultaneously or in parallel and to evaluate them, for example, withthe aid of a computer program which also uses diagnostically significantparameter correlations.

The invention is explained in more detail below by a description of thepreparation of the preferred assay components, the procedure of apreferred embodiment of an assay of the sandwich type and the results ofthe determination of a C-terminal peptide fragment in EDTA plasmas ofcontrol persons and of sepsis, cardiac and cancer patients, obtainedwith the use of such an assay.

Experimental Section

A. Materials and Methods

1. Peptide Syntheses

Derived from the known amino acid sequence of human preproendothelin-1(SEQ ID NO:1), three ranges were selected (Pos. 168-181, 184-203,200-212). In each case supplemented by an N-terminal cysteine residue,these ranges were chemically synthesized as soluble peptides by standardmethods, purified, quality-controlled by means of mass spectrometry andreversed phase HPLC and lyophilized in aliquots (JERINI AG, Berlin,Germany). The amino acid sequences of the peptides are:

Peptide PCT15 (168-181+N-terminal cysteine)

(SEQ ID NO: 4) CRSSEEHLRQTRSET

Peptide PCW14 (200-212+N-terminal cysteine)

(SEQ ID NO: 5) CSRERYVTHNRAHW

Peptide PNR20 (184-203+N-terminal cysteine)

(SEQ ID NO: 6) NSVKSSFHDPKLKGKPSRER

Furthermore, the following peptide was synthesized as a standard forcalibrating the assays:

Standard peptide PSW44 (169-212)

(SEQ ID NO: 7) SSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAHW

2. Conjugation and Immunization

The peptides PCT15 and PCW14 were conjugated with the carrier proteinKLH (keyhole limpet hemocyanine) by means of MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester) (cf. operatinginstructions “NHS-Esters-Maleimide Crosslinkers”, from PIERCE, Rockford,Ill., USA). Sheep were immunized with these conjugates according to thefollowing scheme: each sheep initially received 100 μg of conjugate(stated mass based on the peptide fraction of the conjugate) and then 50μg portions of conjugate every 4 weeks (stated mass based on the peptidefraction of the conjugate). Beginning with the fourth month afterbeginning of the immunization, 700 ml of blood per sheep were takenevery 4 weeks and antiserum was obtained therefrom by centrifuging.Conjugations, immunizations and recovery of antisera were carried out byMicroPharm, Carmarthenshire, UK.

3. Purification of the Antibodies

The peptide-specific antibodies were prepared in a one-step method fromthe antisera which had been recovered beginning with the fourth monthafter immunization.

For this purpose, the peptides PCT15 and PCW14 were first coupled toSulfoLink Gel (cf. operating instruction “SulfoLink Kit”, from PIERCE,Rockford, Ill., USA). In each case 5 mg of peptide per 5 ml of gel wereoffered for coupling.

The affinity purification of peptide-specific antibodies from sheepantisera against both peptides was carried out as follows:

The peptide columns were first washed three times alternately with 10 mleach of elution buffer (50 mM citric acid, pH 2.2) and binding buffer(100 mM sodium phosphate, 0.1% Tween, pH 6.8). 100 ml of the antiserawere filtered with 0.2 μm, and the column material present was added.For this purpose, the gel was quantitatively rinsed from the column with10 ml of binding buffer. The incubation was effected overnight at roomtemperature with swirling. The batches were transferred quantitativelyinto empty columns (NAP 25, Pharmacia, emptied). The runnings werediscarded. The columns were then washed protein-free with 250 ml ofbinding buffer (protein content of the wash eluate <0.02 A280 nm).Elution buffer was added to the washed columns, and 1 ml fractions werecollected. The protein content of each fraction was determined by meansof the BCA method (cf. operating instructions of PIERCE, Rockford, Ill.,USA). Fractions having protein concentrations >0.8 mg/ml were pooled.After protein determination of the pools by means of the BCA method,yields of 97 mg for the anti-PCT15 antibody 0407-pAk and 60 mg for theanti-PCW14 0410-pAk antibody were obtained.

4. Marking

The anti-PCW14 0410-pAk antibody was treated as follows:

500 μl of the purified antibody were rebuffered in 1 ml of 100 mMpotassium phosphate buffer (pH 8.0) according to the operatinginstructions over an NAP-5 gel filtration column (Pharmacia). Theprotein concentration determination of the antibody solution gave avalue of 1.5 mg/ml.

For chemiluminescence marking of the antibody, 10 μl of MA70acridinium-NHS-ester (1 mg/ml; from HOECHST Behring) were added to 67 μlof the antibody solution and incubated for 15 minutes at roomtemperature. Thereafter, 423 μl of 1M glycine were added and incubationwas effected for a further 10 minutes. Thereafter, the marking batch wasrebuffered according to operating instructions over an NAP-5 gelfiltration column (Pharmacia) in 1 ml of mobile phase A 150 mM potassiumphosphate, 100 mM NaCl, pH 7.4) and freed from low molecular weightconstituents. A gel filtration HPLC was carried out for separating offfinal residues of labels not bound to antibodies (column: Waters ProteinPak SW300). The sample was applied and was chromatographed at a flowrate of 1 ml/min with mobile phase A. The wavelengths 280 nm and 368 nmwere measured using a flow photometer. The absorption ratio 368 nm/280nm as a measure of the degree of marking of the antibody was 0.10 at thepeak. The fractions containing monomeric antibodies (retention time 8-10min) were collected, and were collected in 3 ml of 100 mM sodiumphosphate, 150 mM NaC1, 5% bovine serum albumin, 0.1% sodium azide, pH7.4.

5. Coupling

The anti-PCT15 antibody 0407-pAk was treated as follows:

Irradiated 5 ml polystyrene tubes (from Greiner) were coated withpurified antibody as follows: the antibody was diluted to aconcentration of 6.6 μg/ml in 50 mM Tris, 100 mM NaCl, pH 7.8. 300 μl ofthis solution were pipetted into each tube. The tubes were incubated for20 hours at 22° C. The solution was filtered with suction. Each tube wasthen filled with 4.2 ml of 10 mM sodium phosphate, 2% Karion FP, 0.3%bovine serum albumin, pH 6.5. After 20 hours, the solution was filteredwith suction. Finally, the tubes were dried in a vacuum drier.

B. Carrying Out and Evaluating the Immunoassay

An assay buffer of the following composition was prepared: 100 mM sodiumphosphate, 150 mM NaCl, 5% bovine serum albumin (BSA), 0.1% unspecifiedsheep IgG, 0.1% sodium azide, pH 7.4

The above-mentioned chemically synthesized peptide (peptide PSW44) whichcorresponds to the positions 169-212 of preproendothelin-1 serves asstandard material. This was serially diluted in horse normal serum (fromSIGMA). Concentrations according to the weight of the peptide taken wereascribed to the standards thus prepared.

Measuring samples were EDTA plasmas of apparently healthy persons, ofpatients with sepsis and of patients with various cardiovasculardiseases.

50 μl of standards or samples and 200 μl of assay buffer were pipettedinto the test tubes. Incubation was effected for two hours at 22° C.with shaking. Thereafter, washing was effected 4 times with 1 ml of washsolution (0.1% Tween 20) each time per tube and the latter were allowedto drip off. 200 μl of assay buffer, containing 1 million RLU (relativelight units) of the MA70-marked antibody, were then pipetted. Incubationwas effected for two hours at 22° C. with shaking. Thereafter, washingwas effected 4 times with 1 ml of wash solution (0.1% Tween 20) eachtime per tube, the latter were allowed to drip off and thechemiluminescence bound to the tube was measured in a luminometer (fromBERTHOLD, LB952T; base reagents BRAHMS AG).

Using the MultiCalc software (spline fit), the concentrations of thesamples were read from the standard curve.

C. Results

The analyte measurable using the sandwich immunoassay developed(antibody against the positions 168-181 and 200-212) is referred tobelow as C-terminal proendothelin or Ct-proendothelin. A typicalstandard curve for the test developed is shown in FIG. 1. By means ofthe test, it is also possible to determine Ct-proendothelinconcentrations substantially below 50 μg/ml.

In order to examine the question as to whether problems were to beexpected in a measurement of the C-terminal peptide fragment owing toinsufficient stability in a sample or measuring solution, 5 sepsisplasmas were measured in each case fresh and after storage for 12 hoursat room temperature. The results are summarized in FIG. 2. They showthat, after storage for 12 days, the immunoreactivity was virtuallyunchanged at about 93% of the initially measured immunoreactivity. Thisstability detected is a major advantage for diagnostics from points ofview relating to handling.

By means of the test, plasmas of cardiological and sepsis patients weremeasured. The results obtained are shown in FIGS. 3 a and 3 b. For allcardiological clinical pictures investigated, increased values werefound compared with normal controls. Increased values were also foundfor patients with SIRS (systemic inflammatory response syndrome) andseptic conditions. The diagnostic sensitivity (at given 100%specificity, based on healthy controls) increased with the severity ofthe disease: sepsis 32.3%, severe sepsis 65.5% and septic shock 75%.

When the samples were measured using a modified assay in which one ofthe antibodies of the above-mentioned sandwich assay was replaced by anantibody which detected the amino acids 184-203 of preproendothelin-1,substantially identical results were obtained, as expected.

On the other hand, when one of the antibodies used recognized an aminoacid sequence which is localized more closely to the N-terminus of thepreproendothelin (32-52 or 136-148), it was not possible to obtainmeasured values raised compared with healthy persons. This indicatesthat proendothelin as such was not present in the plasma samplesmeasured and is not proteolytically processed only with formation of bigendothelin, but that the C-terminal sequence 93-212 liberated is alsofurther cleaved, it being necessary for at least one such cleavage pointto be present in the range of the amino acids 149-167. The statementapplies to the plasmas of patients with the diseases investigated.However, it cannot be ruled out that, for example, the entire C-terminalfragment 93-212 is retained in other patient groups and its selectivemeasurement can deliver diagnostically relevant results.

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1-19. (canceled)
 20. An isolated antibody which binds specifically to apeptides consisting of amino acids (i) 168-181, (ii) 184-203 or (iii)200-212 of preproendothelin-1 (SEQ ID NO: 1).
 21. The antibody of claim20, wherein said antibody is affinity-purified using (i) peptide168-181, (ii) peptide 184-203 or (iii) peptide 200-212, respectively, ofpreproendothelin-1 (SEQ ID NO: 1).
 22. A kit for, detecting C-terminalfragments of amino acids 168-212 of preproendothelin-1 (SEQ ID NO: 1),said kit comprising (a) a first antibody, selected from antibodies thatspecifically bind to (i) a peptide consisting of amino acids 168-181 or(ii) antibodies that specifically bind to a peptide consisting of aminoacids 184-203 of preproendothelin-1 (SEQ ID NO: 1); and (b) a secondantibody selected from antibodies that specifically bind to a peptideconsisting of amino acids 184-203 or antibodies that specifically bindto a peptide consisting of amino acids 200-212 of preproendothelin-1(SEQ ID NO: 1), wherein said second antibody is different from saidfirst antibody.
 23. The kit of claim 22, wherein one of said first andsecond antibodies is immobilized on the walls of a test tube.
 24. Thekit of claim 22, wherein at least one of said first and secondantibodies has a detectable marker and the other is immobilized orimmobilizable on a solid support.
 25. The kit of claim 22, furthercomprising (c) a standard peptide comprising at least amino acids168-203 or 168-212 of preproendothelin-1 (SEQ ID NO: 1).
 26. Theisolated antibody of claim 20, wherein said first antibody specificallybinds to a peptide consisting of amino acids 168-181 ofpreproendothelin-1 (SEQ ID NO: 1).
 27. The isolated antibody of claim20, wherein said antibody specifically binds to a peptide consisting ofamino acids 184-203 of preproendothelin-1 (SEQ ID NO: 1).
 28. Theisolated antibody of claim 20, wherein said antibody specifically bindsto a peptide consisting of amino acids 200-212 of preproendothelin-1(SEQ ID NO: 1).
 29. The kit of claim 22, comprising (a) a first antibodythat specifically binds to a peptide consisting of amino acids 168-181;and (b) a second antibody that specifically binds to a peptideconsisting of amino acids 184-203 of preproendothelin-1 (SEQ ID NO: 1).30. The kit of claim 29, further comprising (c) a standard peptidecomprising at amino acids 168-203 of preproendothelin-1 (SEQ ID NO: 1).31. The kit of claim 22, comprising (a) a first antibody thatspecifically binds to a peptide consisting of amino acids 168-181; and(b) a second antibody that specifically binds to a peptide consisting ofamino acids 200-212 of preproendothelin-1 (SEQ ID NO: 1).
 32. The kit ofclaim 31, further comprising (c) a standard peptide comprising at leastamino acids 168-212 of preproendothelin-1 (SEQ ID NO: 1).
 33. The kit ofclaim 22, comprising (a) a first antibody that specifically binds to apeptide consisting of amino acids 184-203; and (b) a second antibodythat specifically binds to a peptide consisting of amino acids 200-212of preproendothelin-1 (SEQ ID NO: 1).
 34. The kit of claim 33, furthercomprising (c) a standard peptide comprising at least amino acids168-212 of preproendothelin-1 (SEQ ID NO: 1).